The identity of the discriminator base has an impact on CCA addition

Abstract

CCA-adding enzymes synthesize and maintain the C-C-A sequence at the tRNA 3'-end, generating the attachment site for amino acids. While tRNAs are the most prominent substrates for this polymerase, CCA additions on non-tRNA transcripts are described as well. To identify general features for substrate requirement, a pool of randomized transcripts was incubated with the human CCA-adding enzyme. Most of the RNAs accepted for CCA addition carry an acceptor stem-like terminal structure, consistent with tRNA as the main substrate group for this enzyme. While these RNAs show no sequence conservation, the position upstream of the CCA end was in most cases represented by an adenosine residue. In tRNA, this position is described as discriminator base, an important identity element for correct aminoacylation. Mutational analysis of the impact of the discriminator identity on CCA addition revealed that purine bases (with a preference for adenosine) are strongly favoured over pyrimidines. Furthermore, depending on the tRNA context, a cytosine discriminator can cause a dramatic number of misincorporations during CCA addition. The data correlate with a high frequency of adenosine residues at the discriminator position observed in vivo. Originally identified as a prominent identity element for aminoacylation, this position represents a likewise important element for efficient and accurate CCA addition.

Figure 1.

Substrates for CCA addition. Left: Randomized RNA sequences were incubated in presence (+) and absence (-) of the human CCA-adding enzyme. The enzyme incorporates nucleotides in some transcripts, leading to a reduced electrophoretic mobility, visible as a smear (boxed) above the main band of the RNA pool. Right: Sequences of 47 individual RNA substrates retrieved from the shifted transcripts. Due to the construction of the library for efficient T7 transcription, all sequences start with GG. Nucleotides incorporated by the CCA-adding enzyme are shown on the right. The base located immediately upstream of the added nucleotides (corresponding to the tRNA discriminator position 73) is shown separately. Here, a strong excess of adenosine (red) is visible. While most of the clones carry a complete or partial CCA end, some show additional C residues incorporated.

Figure 2.

Individual candidate transcripts are true substrates for CCA addition. From the list of candidates presented in Figure 1, 13 RNA molecules were tested individually with the CCA-adding enzyme (+). The negative control represents the transcripts incubated without enzyme. All candidates show nucleotide additions with varying efficiency, ranging from the incorporation of a single nucleotide (partial CCA end, #22) to the addition of three or more residues. The secondary structure models show that most of the candidates carry base-paired 5′- and 3′-ends with 3′-terminal nucleotide overhangs, corresponding to an acceptor stem-like structure. Yet, also single-stranded 3′-ends are tolerated for nucleotide addition, although at a rather low efficiency (#5, #25, #43).

Figure 3.

Competition studies with candidates and tRNAs. Substrate candidates #4, #6 and #12 were incubated as radioactively labelled transcripts with increasing concentrations of unlabelled tRNA as competitor. While the yeast tRNA^Phe (F) shows an efficient competition leading to reduced NTP incorporation into the candidates, the human mitochondrial tRNA^Tyr (Y) is competing less efficiently. The nucleotide addition in the presence of intact tRNA indicates that the candidates are indeed competent substrates, although at a lower efficiency compared to tRNA^Phe.

Figure 4.

The discriminator base affects CCA addition. From 5 min to 4 h of incubation with human CCA-adding enzyme, the incorporation of CCA termini in the human mitochondrial tRNA^Tyr was monitored by the appearance of shifted bands in the polyacrylamide gels. The wild-type transcript with A73 shows the fastest addition, with a complete turnover after 1 h. Transcripts with G73 or U73 show a complete CCA addition after 2 or 4 h, respectively. While the reaction stops after addition of three residues to these substrates, the tRNA ending with C73 shows further band shifts, indicating the incorporation of additional nucleotides.

Figure 5.

Competition study with tRNA variants carrying different discriminator bases. (A) Equimolar amounts of radioactively labelled transcripts of human mitochondrial tRNA^Tyr with A73, G73, C73 and U73 were incubated with the human CCA-adding enzyme in a time series. At the indicated time points, reaction products were separated by denaturing gel electrophoresis. The reduced migration of the signal bands indicates the addition of one to three nucleotides, corresponding to the CCA terminus. Lower panel: quantitative analysis of individual nucleotide additions. After the first time point, a clear preference for tRNA carrying purine discriminators is detectable. About 40% of the analysed clones carry either G or A at position 73. Over the whole time course, tRNAs with A73 or G73 represent the preferred substrates for CCA addition, while transcripts ending with C73 or U73 show a dramatically reduced CCA addition. (B) Competition experiment with human tRNA^Pro. The experiment was conducted according to (A). tRNA^Pro with A73 is a much better substrate for CCA addition than the native transcript with C73. Yet, CCA incorporation in this transcript is as efficient as for tRNA^Pro with G73. While again a purine (adenosine) discriminator is highly preferred, the wild-type tRNA^Pro with a pyrimidine discriminator is a much better substrate than in the case of tRNA^Tyr, indicating that the context of tRNA^Pro compensates for the non-optimal discriminator base.

Figure 6.

Erroneous nucleotide additions. tRNA substrates isolated in the competition experiment were analysed for nucleotide misincorporation catalysed by the CCA-adding enzyme. Whereas tRNA^Tyr (blue bars) with A, G or U residues at position 73 showed almost exclusively correct CCA additions, ∼70% of the corresponding transcripts with C73 carried misincorporations and extra nucleotides. Order and number of incorporated C and A residues showed a strong variability, leading to sequences with up to five added nucleotides in varying combinations of C and A. tRNA^Pro (green bars), on the other hand, showed only slightly increased misincorporation, if the discriminator base is a cytidine. Again, the structural context of this tRNA seems to compensate for the unfavourable C73 position.